Fluorescent probes for metal ions such as Ca(ll) have proven to be tremendously valuable tools for biological research, particularly in neuroscience where metals play roles as chemical messengers. "On- fluorescence," where the probe lights up or changes color, is greatly preferred to "off-fluorescence", where fluorescence is quenched by the analyte. The ideal probe would be ratiometric, showing off-fluorescence at one wavelength and proportionate increase in fluorescence at another wavelength. Many biologically interesting metal ions that are paramagnetic including Cu(ll), Fe(ll), Mn(ll), Ni(ll), Co(ll) and others can only be studied by off-fluorescence using known probes because their unpaired electrons provide a mechanism to quench fluorescence. The goal of this project is to develop a new approach that allows on-fluorescence detection of paramagnetic metal ions. In preliminary experiments, ratiometric fluorescence titration of Mn(ll) was observed under solution conditions similar to physiological. The metal Mn(ll) is used widely as a tool in manganese-enhanced magnetic resonance imaging (MEMRI) studies of brain function in mice. Mn(ll) passes calcium channels without blocking them, making it a unique and valuable tool for neurological research. However, it has not yet been possible to correlate Mn(ll) concentrations directly with observed contrast as no adequate tool is available to do so. The probes described in this proposal would thus provide needed confirmation of assumptions made in MEMRI research. Development of a fluorescence assay to complement MEMRI techniques would allow the test Mn(ll) uptake and release in the brain, test for regional localization within the brain, and allow identification of tissue-specific uptake. Thus, the overall goal of this R21 application is to observe Mn(ll) distribution in mouse brain by fluorescence, providing a powerful new tool for neurological research, and demonstrating a useful new strategy that can be applied broadly in biomedical research. [unreadable] [unreadable]